Competitive binding immunoassays for quantitatively measuring the presence of physiologically active compounds (ligands) are based on the competition between a ligand in a test sample and a labeled reagent, referred to as a tracer, for a limited number of binding sites on antibodies or receptors specific to the ligand and the tracer. The concentration of ligand in a sample to be assayed determines the amount of tracer or label that will specifically bind to an antibody. By measuring the amount of tracer-antibody complex a quantitative determination of the amount of ligand in the test sample is provided. When necessary, modification of such ligand to prepare an immunogen should take into account the effect on the structural specificity of the antibody. That is, in choosing a site on a ligand for conjugation to a carrier such as protein, the selected site is chosen so that administration of the resulting immunogen will provide antibodies which will recognize the original ligand. Furthermore, not only must the antibody recognize the original ligand, but significant characteristics of the ligand portion of the immunogen must remain so that the antibody produced after administration of the immunogen may distinguish compounds closely related to the ligand from other compounds which may also be present in the patient sample. In addition, the antibodies should have high binding constants.
Also, the tracer must effectively compete with the ligand for antibody binding in a reproducible manner and provide for significant changes in the measured signal with small changes in the concentration of the ligand over the concentration range of interest.
Other considerations for an immunoassay method are that it is not affected by materials present in the sample to be assayed, an easily determinable signal is obtained, the tracer, standards and antibodies have good storage life and are stable under the assay conditions. Also, the tracer and standards must be readily recognizable by the antibodies for the ligand.
ddI (2',3'-dideoxyinosine), which can be represented by the formula: ##STR1## has been shown to be an effective drug in the treatment of Acquired Immunodeficiency Syndrome (AIDS). As with any drug, and especially one having serious side effects at high dosage levels, such as ddI, the formal establishment of a therapeutic range for a patient being treated with the drug is necessary. The recognized method for measuring ddI plasma levels employs high performance liquid chromatography (HPLC) which is a sensitive and reasonably precise technique. However, although HPLC can be used for measuring ddI concentrations in noninfective samples and in HIV-positive samples, the HPLC method is not practical for the routine monitoring of ddI in HIV-positive samples due to the disadvantages of a long analysis time, elaborate sample preparation requirements, including complicated solid phase extraction, a relatively large sample size (250 .mu.l-3,000 .mu.l) and interference by body fluid components in the sample. A particular problem in using HPLC to monitor ddI levels in pediatric patients is the difficulty in obtaining samples of sufficient volume. Also, HPLC is subject to interference from endogenous compounds.
The development of an analytical method for measuring ddI levels in a patient being treated with ddI which could minimize sample manipulation, employ disposable equipment as much as possible, reduce the sample size needed and shorten the length of the assay time is therefore needed. It is preferred that such a method not be subject to interference from endogenous compounds, or hemolysis or typically prescribed drugs.